Context-Dependent Action of Scc4 Reinforces Control of the Type III Secretion System.

Chlamydia trachomatis Scc4 (formerly CT663) engages the transcription machinery and the pathogenic type III secretion system (T3SS). Both machines are required for Chlamydia infection. These requirements and the limited ability for genetic manipulation in Chlamydia have hampered dissection of Scc4's contributions. Here, by developing bacterial systems that permit the controlled expression and stable maintenance of Scc4, we assess Scc4's effects on chlamydial growth phenotype, secretion, and the patterns of T3SS gene expression. Expressing Scc4 in Escherichia coli lacking a T3SS injectisome causes a growth defect. This deficiency is rescued by overexpressing the ß-subunit of RNA polymerase (RNAP) or by exploiting sigma 70 (σ70) (homologous to chlamydial σ66) mutants that strengthen the interaction between σ70 region 4 and the ß-flap, confirming Scc4's distinction as a module of RNAP holoenzyme capable of modulating transcription. Yersinia pestis expressing Scc4 sustains a functional T3SS, through which CopN secretion is boosted by cooption of Scc4 and Scc1. Finally, conditional expression of Scc4 in C. trachomatis results in fast expansion of the Chlamydia-containing vacuole and accelerated chlamydial development, coupled to selective up- or downregulation of gene expression from different T3SS genes. This work reveals, for the first time, the context-dependent action of Scc4 linking it to diverse protein networks in bacteria. It establishes that Scc4, when overexpressed, exerts incredible effects on chlamydial development by reinforcing control of the T3SS.IMPORTANCE The T3SS is a key virulence factor required for C. trachomatis infection. The control of the T3SS has not been well studied in this obligate intracellular pathogen. Here, we show that Scc4 plays a major role for precise control of the pathogenic T3SS at the levels of gene expression and effector secretion through genetically separable protein networks, allowing a fast adaptive mode of C. trachomatis development during infection in human epithelial cells.

Mutually constructive roles of Ail and LPS in Yersinia pestis serum survival.

The outer membrane is a key virulence determinant of gram-negative bacteria. In Yersinia pestis, the deadly agent that causes plague, the protein Ail and lipopolysaccharide (LPS)6 enhance lethality by promoting resistance to human innate immunity and antibiotics, enabling bacteria to proliferate in the human host. Their functions are highly coordinated. Here we describe how they cooperate to promote pathogenesis. Using a multidisciplinary approach, we identify mutually constructive interactions between Ail and LPS that produce an extended conformation of Ail at the membrane surface, cause thickening and rigidification of the LPS membrane, and collectively promote Y. pestis survival in human serum, antibiotic resistance, and cell envelope integrity. The results highlight the importance of the Ail-LPS assembly as an organized whole, rather than its individual components, and provide a handle for targeting Y. pestis pathogenesis.

Chromosomally-Encoded Yersinia pestis Type III Secretion Effector Proteins Promote Infection in Cells and in Mice.

Yersinia pestis, the causative agent of plague, possesses a number of virulence mechanisms that allows it to survive and proliferate during its interaction with the host. To discover additional infection-specific Y. pestis factors, a transposon site hybridization (TraSH)-based genome-wide screen was employed to identify genomic regions required for its survival during cellular infection. In addition to several well-characterized infection-specific genes, this screen identified three chromosomal genes (y3397, y3399, and y3400), located in an apparent operon, that promoted successful infection. Each of these genes is predicted to encode a leucine-rich repeat family protein with or without an associated ubiquitin E3 ligase domain. These genes were designated Yersinia leucine-rich repeat gene A (ylrA), B (ylrB), and C (ylrC). Engineered strains with deletions of y3397 (ylrC), y3399 (ylrB), or y3400 (ylrA), exhibited infection defects both in cultured cells and in the mouse. C-terminal FLAG-tagged YlrA, YlrB, and YlrC were secreted by Y. pestis in the absence but not the presence of extracellular calcium and deletions of the DNA sequences encoding the predicted N-terminal type III secretion signals of YlrA, YlrB, and YlrC prevented their secretion, indicating that these proteins are substrates of the type III secretion system (T3SS). Further strengthening the connection with the T3SS, YlrB was readily translocated into HeLa cells and expression of the YlrA and YlrC proteins in yeast inhibited yeast growth, indicating that these proteins may function as anti-host T3S effector proteins.

The YscE/YscG chaperone and YscF N-terminal sequences target YscF to the Yersinia pestis type III secretion apparatus.

The needle structures of type III secretion (T3S) systems are formed by the secretion and polymerization of a needle subunit protein, YscF in Yersinia pestis. A subset of T3S systems employ unique heterodimeric chaperones, YscE and YscG in Y. pestis, to prevent the polymerization of needle subunits within the bacterial cell. We demonstrate that the YscE/YscG chaperone is also required for stable YscF expression and for secretion of YscF. Overexpression of a functional maltose-binding protein (MBP)-YscG hybrid protein stabilized cytoplasmic YscF but YscF was not secreted in the absence of YscE. Furthermore, a YscE mutant protein was identified that functioned with YscG to stabilize cytosolic YscF; however, YscF was not secreted. These findings confirm a role for the YscE/YscG chaperone in YscF secretion and suggest that YscE may have a specific role in this process. Recent studies have shown that YscF deleted of its N-terminal 15 residues is still secreted and functional, suggesting that YscF may not require an N-terminal secretion signal. However, we demonstrate that YscF contains an N-terminal secretion signal and that a functional N-terminal signal is required for YscF secretion.

ExsA and LcrF recognize similar consensus binding sites, but differences in their oligomeric state influence interactions with promoter DNA.

ExsA activates type III secretion system (T3SS) gene expression in Pseudomonas aeruginosa and is a member of the AraC family of transcriptional regulators. AraC proteins contain two helix-turn-helix (HTH) DNA binding motifs. One helix from each HTH motif inserts into the major groove of the DNA to make base-specific contacts with the promoter region. The amino acids that comprise the HTH motifs of ExsA are nearly identical to those in LcrF/VirF, the activators of T3SS gene expression in the pathogenic yersiniae. In this study, we tested the hypothesis that ExsA/LcrF/VirF recognize a common nucleotide sequence. We report that Yersinia pestis LcrF binds to and activates transcription of ExsA-dependent promoters in P. aeruginosa and that plasmid-expressed ExsA complements a Y. pestis lcrF mutant for T3SS gene expression. Mutations that disrupt the ExsA consensus binding sites in both P. aeruginosa and Y. pestis T3SS promoters prevent activation by ExsA and LcrF. Our combined data demonstrate that ExsA and LcrF recognize a common nucleotide sequence. Nevertheless, the DNA binding properties of ExsA and LcrF are distinct. Whereas two ExsA monomers are sequentially recruited to the promoter region, LcrF binds to promoter DNA as a preformed dimer and has a higher capacity to bend DNA. An LcrF mutant defective for dimerization bound promoter DNA with properties similar to ExsA. Finally, we demonstrate that the activators of T3SS gene expression from Photorhabdus luminescens, Aeromonas hydrophila, and Vibrio parahaemolyticus are also sensitive to mutations that disrupt the ExsA consensus binding site.

Na+/H+ antiport is essential for Yersinia pestis virulence.

Na(+)/H(+) antiporters are ubiquitous membrane proteins that play a central role in the ion homeostasis of cells. In this study, we examined the possible role of Na(+)/H(+) antiport in Yersinia pestis virulence and found that Y. pestis strains lacking the major Na(+)/H(+) antiporters, NhaA and NhaB, are completely attenuated in an in vivo model of plague. The Y. pestis derivative strain lacking the nhaA and nhaB genes showed markedly decreased survival in blood and blood serum ex vivo. Complementation of either nhaA or nhaB in trans restored the survival of the Y. pestis nhaA nhaB double deletion mutant in blood. The nhaA nhaB double deletion mutant also showed inhibited growth in an artificial serum medium, Opti-MEM, and a rich LB-based medium with Na(+) levels and pH values similar to those for blood. Taken together, these data strongly suggest that intact Na(+)/H(+) antiport is indispensable for the survival of Y. pestis in the bloodstreams of infected animals and thus might be regarded as a promising noncanonical drug target for infections caused by Y. pestis and possibly for those caused by other blood-borne bacterial pathogens.